Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

Abstract

Small heat-shock proteins (sHSP) are molecular chaperones that bind and sequester partially and globally unfolded states of their client proteins. Of paramount importance to their physiological roles is the assembly into large oligomers, which for mammalian sHSP are polydisperse and undergo subunit exchange. The flexibility and dynamic nature of these oligomers mediates functional regulation by phosphorylation and underpins the deleterious effects of disease-linked mutations. Previously, we discovered that the archaeal Hsp16.5, which natively forms ordered and symmetric 24-subunit oligomers, can be engineered to transition to an ordered and symmetric 48-subunit oligomer by insertion of a peptide from human HspB1 (Hsp27) at the junction of the N-terminal and -crystallin domains. Here, we carried out a detailed analysis of the determinants of Hsp16.5 oligomeric plasticity by altering the sequence and length of the inserted peptide. Utilizing light scattering, blue native gel electrophoresis, native mass spectrometry and electron microscopy, we uncovered the existence of an array of oligomeric states (30 to 38 subunits) that can be populated as a consequence of different insertions. These oligomers are intermediate states on the assembly pathway of the 48-subunit oligomer as two of the variants can concurrently form 24-subunit or 30-38 subunit polydisperse oligomers. Polydisperse Hsp16.5 oligomers displayed higher affinity to a model client protein consistent with a general mechanism for recognition and binding that involves increased access of the hydrophobic N-terminal region. Our findings, which integrate structural and functional analyses from evolutionarily-distant sHSP, support a model wherein the modular architecture of these proteins encodes motifs of oligomer polydispersity, dissociation and expansion to achieve functional diversity and regulation.